The TCR-CD3 complex, CD4, and CD28 are vital checkpoint molecules that allow T cells to survey for antigens and activation induced molecules on the surfaces of antigen presenting cells (APCs). They then transfer this antigen- and APC-specific information to the T cell's intracellular signaling apparatus. Ultimately, this informatio directs the T cell fate decisions that drive development, activation, differentiation, and the execution of effector functions. The fundamental importance of these molecules to immune surveillance and human health has resulted in several studies regarding their structure and function over the past 25+ years. As a result, much is now known about their individual structures, their interactions with their respective ligands in isolation, and the signaling cascads that result from these interactions. But, we have yet to determine how they fit and work together as components of the molecular machinery that drives T cell fate decisions and this has prevented us from understanding how this molecular machinery, as a whole, executes its functions. Further, this lack of knowledge has handicapped our ability to strategically target this molecular machinery with reagents designed to either enhance responses to vaccines, tumor, or pathogens, or to attenuate responses to transplants or auto-antigens. Our goal is to deconstruct, understand, and ultimately manipulate the form and function of this complex macromolecular machine. To this end, we will: 1) determine how antigen-specific information is relayed across the membrane by the TCR to the intracellular signaling domains of the CD3 subunits; 2) determine how these signaling subunits are positioned in close proximity with the enzymes that modify them; and 3) determine how the surfaces that stabilize the architecture of this higher order machinery influence CD4+ T cell fate decisions in vivo. We have developed a novel experimental platform that combines classic biochemical and molecular biology techniques with modern polycistronic retroviral systems, kinase- based dimerization assays, and live cell fluorescent video imaging (including the use of total internal reflection fluorescence microscopy (TIRFM) and Forster Resonance Energy Transfer (FRET)), to take a highly controlled reductionist approach to addressing Aim 1 and 2. In addition, we are building novel mouse model systems to accomplish Aim 3. Specifically we will study the surfaces that we have already identified as mediating TCR- CD3 complex stability and TCR dimerization to determine how these interactions, which are at the core of the higher-order macromolecular machinery that transfers pMHC-specific information from the outside to the inside of a cell, influence T cell fate decisions in vivo. Altogether, these experiments will yield important insights into the molecular mechanisms that underlie CD4+ T cell fate decisions and identify potential targets for the development of translational immune-modulating reagents. PUBLIC HEALTH RELEVANCE: This proposal is focused on increasing our understanding of three critical but poorly understood aspects of T cell biology: (i) how information regarding the quantity and quality of antigen is transferred from the outside to the inside of a T cell; (ii) how information about the context in which the antigen occurs on the surface of an APC (i.e. the APC activation state) is transferred from the outside to the inside of a T cell; (iii) how these mechanisms impact T cell fate decisions (e.g. development, activation, differentiation, execution of effector functions). An intended consequence of these studies will be the identification and characterization of potential targets for translational immune-modulatory reagents.